Diagnosis of glioblastoma multiform (GBM) with MRI lacks molecular information and requires a biopsy for pathologic confirmation. The EGFRvIII, is a constitutively active mutant of the EGF receptor, identified in a high percentage of brain cancers and associated with increased invasiveness and resistance, making it a good target to improve imaging and diagnosis. The present study shows that conjugation of near-infrared quantum dot (Qd800) to an anti-EGFRvIII single domain antibody, made of the variable region with an extra cysteine for site-specific conjugation (EG2-Cys), increased its internalization in U87MG-EGFRvIII cells in vitro compared to Qd800 conjugated with the Fc region of the antibody (EG2-hFc) or unconjugated. EG2-Cys also improved the contrast in Near-Infrared Imaging of mice bearing orthotopic glioblastoma. The increased accumulation was confirmed by fluorescence microscopy of brain sections. The specificity of EG2-Cys in brain tumor expressing the EGFRvIII mutant receptor may provide an accurate less invasive diagnosis and determine the level of tumor aggressiveness and resistance.